Background: Ghrelin was initially recognized as an endogenous ligand of growth hormone secretagogue receptor and was implicated in the regulation of food intake, and promoting weight gain. Ghrelin has been shown to improve cardiac function in patients suffering from heart failure (HF) though various mechanisms. The aim of the review is to summarize the main findings in this field, with the purpose of promoting further studies on the role of ghrelin on the cardiovascular system. Materials and Methods: All publications describing trials, systematic reviews, meta-analyses and review papers published within 1999-2014 of ghrelin in animal models of HF were sought through electronic and manual searches. Results: The literature searches identified 126 references and ten trials meeting the inclusion criteria were included in this review. All studies were carried out on male rats and experimental model of HF. Ghrelin has been shown to reduce mortality, increase appetite and body weight, and was found to improve the cardiac function parameters. Review found deficient information about adverse effects of ghrelin. Ghrelin exerts cardioprotective effects through modulation of sympathetic nervous system, inhibiting autophagy, antiinflammatory effects and protection against ischemia/reperfusion injury. Conclusion: Ghrelin seems to have a beneficial effect in rat models of HF and can offer an effective therapeutic target for improving outcome in HF.

The ultimate outcome of most of the cardiovascular diseases is chronic heart failure (CHF) and it is a major cause of morbidity and mortality in patients with cardiovascular diseases. [1] Ghrelin, a peptide of 28 amino acid polypeptide, was first reported in 1999 by Kojima in rat and human stomachs. [2] Ghrelin, was initially recognized as an endogenous ligand of growth hormone secretagogue receptor (GHS-R) and was implicated in the regulation of food intake, and promoting weight gain. [2],[3],[4],[5],[6],[7]

Ghrelin also has a role in modulation of gastrointestinal, cardiovascular, pulmonary and immune functions, cell proliferation/apoptosis. [8] There is a widespread distribution of ghrelin and its receptors in the cardiovascular tissues. The protective effects of ghrelin on heart are mediated through direct effects on the heart and blood vessel and through. Its growth-hormone-releasing effect. Cardioprotective effects of ghrelin have been demonstrated on heart failure (HF) models and exploratory human clinical studies. [9],[10],[11],[12],[13] Elevated levels of ghrelin have been found in patients with HF. [14] Various cardioprotective effects of ghrelin have been reported which includes reduction of peripheral vascular resistance, [15],[16] improvement in myocardial contractility and antiinflammatory effects. [17] Clinical studies have reported that exogenous administration of ghrelin decreases muscle wasting, improves exercise capacity, improves left ventricular (LV) and endothelial function, increase myocardial contractility, dilate peripheral blood vessels, constricts the coronary arteries, reduces blood pressure, inhibits cardiomyocyte apoptosis, inhibit sympathetic nerve activity (SNA) and protects from HF induced by myocardial infarction. [8],[18],[19],[20],[21],[22] So also, ghrelin may have other cardiovascular protective effects such as prevention of atherosclerosis as well as protection from ischemia and reperfusion injury. [18],[19],[23],[24] Importantly, administration of ghrelin has been demonstrated to improve the cardiac function and prognosis in patients suffering from end-stage CHF. [13]In vitro, ghrelin decreases inotropism [13],[14] and lusitropism. [14] Ghrelin administration also benefits the patients suffering from cardiac cachexia in CHF by inducing a positive energy balance. [25]

Thus, ghrelin seems to exert cardioprotective effects through its broadspectrum effects and the researches points that ghrelin can be a new therapy for HF and other cardiovascular diseases. However, exact effects of ghrelin on the cardiovascular system are inconclusive at present. The purpose of this review is to comprehensively appraise the current literature regarding effectiveness of ghrelin on weight gain and cardiovascular outcomes in experimental rat models of HF and to provide molecular insights that corroborate physiological cardiovascular protection by ghrelin. The aim of the review is to summarize the main findings in this field, with the purpose of promoting further researches on the role of ghrelin on cardiovascular system.

Criteria for considering studies of this review

Types of studies

Randomized controlled trials (RCTs) and nonrandomized studies which evaluated the effects of ghrelin in experimental rat models of CHF. Studies published only in abstract forms or nonpeer reviewed journals were excluded.

Types of participants

Trials on rat models of HF were included.

Types of interventions

Studies were included in which experimental intervention was done with any ghrelin at any dose, any analog, any route, administered as single agents/combination therapies and in fixed/stepped/titrated doses.

All publications describing controlled trials of ghrelin in rat models of HF were sought through electronic searches on Cochrane Central Register of Controlled Trials on Cochrane Library, MEDLINE (1999 to May 2014), EMBASE (1999 to May 2014), CINAHL (1999 to May 2014), AMED (1999 to May 2014). Digital dissertations. Conference proceedings were searched on Web of Science. Additional studies were located in Health Technology Assessment and Database of Abstracts of Reviews of Effects. Bibliographies of all papers were searched for further trials. No restrictions regarding language of publication were imposed. Search was focused on randomized and controlled trials, systematic reviews, meta-analyses and review papers published within 1999-2014. To avoid missing of studies in search strategy, consideration was given to spelling of terms used in different countries. If any data were found insufficient, authors were contacted through E-mail. Book chapters and editorials were also scanned. Manufacturers of ghrelin preparations experts and authors on the subject were contacted through emails and asked to contribute published and unpublished material. Hand searches were conducted for journals and conference proceedings.

Data collection

Two authors independently assessed all potentially relevant trials according to prespecified selection criteria with an emphasis on selecting RCTs and nonrandomized trials. References identified by electronic search strategy were screened by title and abstract to determine eligibility for inclusion in this systematic review and studies that appeared irrelevant were discarded. Full text of potentially eligible studies were retrieved (and translated into English where required) and two reviewers then independently determined study eligibility using a standardized inclusion form. Details of the studies were extracted and included in characteristics of studies table [Table 1]. Any disagreement about eligibility of study was resolved by discussion. Furthermore, a third reviewer did an independent review to settle any difference of opinion between two primary reviewers.

Literature search identified 126 references that described 57 potentially relevant trials, including two unpublished studies and one conference abstract. Twenty-six trials have been carried out in animal such as rats, mice, pigs and rabbits. There were only three human clinical trials evaluating the role of ghrelin in patients with CHF. Ten trials met the inclusion criteria and were included in this review. Three trials were carried out each in Germany, Japan and China and one trial New Zealand. Sample size ranged from 36 to 121 rats with a total of 382 rats recruited in ten studies. Six studies were done on Sprague-Dawley rats, one on Wistar rats, two on C57BL/6J rats and one on Ghrelin-knockout mice. All studies were done on male rats weighing between 200 and 340 g and experimental model of HF was created by ligation of coronary artery except in three studies. In two studies HF was induced by isoprenaline and in one study, it was induced by doxorubicin. Controls were subjected to sham operation of a thoracotomy and cardiac exposure without ligation of coronary artery or by injection of normal saline. Eight trials administered ghrelin subcutaneously and one trial each administered ghrelin orally and intraperitoneally. Trial periods lasted for as less as 2 days and as long as 28 days. In all trials, ghrelin was not used as an adjunct to conventional treatment for HF. Two trials compared two analogs of ghrelin BIM-28125 and BIM-28131 in two different doses. Review found deficient information about effect of ghrelin on adverse effects of ghrelin needed in recommendation for adoption of this therapy in treating CHF. Characteristics of included studies are as shown in [Table 1].

Results of search

Effect on mortality

Mortality was reported in four studies. The mortality was significantly lower after administration of ghrelin [26],[27] and hexarelin group. [28] However; mortality did not differ between a single oral dose of hexarelin and vehicle group. [29] Increasing the dose of ghrelin from 1 nmol/kg/d to 10 nmol/kg/d significantly reduces the mortality in HF [Table 2]. [26]

In Sandra's study, [30] food intake per day got lesser during both the treatment periods (treatment period 1: day 28-41, treatment period 2: 42-56). During day 28-41, food intake increased in all high dose of human ghrelin as well as with both the analogues BIM-28131 and BIM-28125 and also with BIM-28131 in low dose when compared to placebo. In second treatment period (from day 42); higher food intake was seen in all the group, which were on ghrelin all groups given ghrelin compounds had a higher food intake than placebo.

Effects of ghrelin on body weight and somatotropic functions

Effect on body weight

Body weight was reported in three studies. In Nagaya's study, the greater increase in body weights was seen in HF rats with ghrelin than with placebo. [9] In Sandra's study; after 2 weeks of administration of compounds of ghrelin or placebo, rats that received low dose BIM-28131 or high dose human ghrelin, BIM 28131 or BIM 28125 demonstrated a significantly higher weight gain than rats that were on placebo and also rats with sham surgery. Insignificant differences in weight gain were seen with a higher dose of BIM 28131 as compared to the low dose. [30] In Mao's study, body weight was lower in hexalin group as compared to Sham group [Table 3]. [29]

Tibial length and gastrocnemius muscle weight were significantly increased after administration of ghrelin as compared with those given placebo. [9] In Sandra's study, tibial length was similar between the groups and significantly different than the respective placebo group. [30] LV weight/tibial length was significantly higher in CHF rats on ghrelin than in CHF rats on placebo while right ventricle (RV) weight/tibial length was significantly higher in CHF rats treated with placebo than CHF rats treated with ghrelin. [9] The ratio of weight of the gastrocnemius muscle to tibial length and muscle protein content to tibial length were higher significantly in sham rats on ghrelin than in CHF rats on ghrelin. [9] The septum weights were higher in all compounds of ghrelin, except for low dose. [30]

Effect on expression of muscle proteins

Expression of MAFbx was down-regulated to normal levels by low dose BIM-28125 and high dose BIM-28131 while expression of MuRF-1 was reduced to sham levels by all ghrelin compounds except high dose BIM-28125 group. [30] After induction of HF in rats, expression of myostatin protein in the gastrocnemius muscle was significantly down regulated in animals administered with human ghrelin or ghrelin analogs in low and high doses (except high dose BIM-28125) when compared to rats on placebo. [31] Plasma ET-1 levels were reduced significantly in ghrelin group when compared to the ISO group, while the expression of myocardial ET-1 mRNA was significantly higher in ISO group when compared with the control group [Table 4]. [27]

Lin's study showed that administration with ISO increased HR significantly as compared to the control group, which was not improved by administration of ghrelin. [26] In Daryl's and Nagaya's study, there was an insignificant difference in HR between ghrelin and other groups. [9],[27] Effect of chronic administration of ghrelin on HR regulation is similar that observed during acute ghrelin infusion. [34] Higher doses of ghrelin lead to more reductions in HR when compared to lower doses [Table 5]. [26],[32]

Systemic vascular resistance [9],[27] and mean right atrial pressure were significantly and insignificantly lower respectively [9] in HF rats on ghrelin than on placebo. Values of cardiac output, [9],[27],[28],[29] stroke volume, [9],[27] ejection fraction, [27],[28],[29],[30] LVdP/dtmax [9],[26],[27] was higher in CHF rats treated with ghrelin/hexarelin than given placebo. [9],[26],[27] LV diastolic dimension was decreased in Nagaya's study, [9] significantly increased in Akashi's study [32] and unaltered in Pei's study. [33] Diastolic thickness of noninfarcted posterior wall, [9] LV fractional shortening [9],[29] and shortening velocity [9] was higher in CHF on ghrelin than on placebo. Decrease in HR, end-diastolic dimension, ejection fraction and left ventricle fractional shortening after exposure to doxorubicin was not seen in mice treated with desacyl ghrelin. [33]

Discussion

In this review we have compiled the results of eight studies assessing the effect of ghrelin on somatotrophic function, expression of muscle proteins and cardiovascular system on rat experimental model of HF. Ghrelin reduces mortality, promotes weight gain and plays a crucial role in protecting heart function. [9],[15],[26],[27],[30],[33] Ghrelin acts through the GHS-R 1a which is expressed in many areas of the central nervous system (CNS), where it arbitrates appetite and adiposity. [35] Ghrelin is the only known peripheral orexigenic hormone, which plays a short term regulatory role in energy balance by inducing appetite and a long-term regulatory role in energy balance by promoting weight gain and adiposity. [36]

Cardiovascular effects

The high expression of ghrelin and GHSR 1a in the heart, and large vessels provide evidence of its cardiac actions. Administration of ghrelin improves cardiac performance in rat models of HF, as indicated by increases in cardiac output, stroke volume, LV dP/dtmax, and LV fractional shortening and by increases in fractional cell shortening and shortening velocity of isolated myocytes. Activating GHS-R 1a might be beneficial for cardioprotection, although other mechanism may also be involved. [29] The myocardium of CHF patients shows an impaired production of ghrelin that causes a compensatory increase in the expression of GHS-R 1a. [37] Elevated levels of ghrelin have been found in patients with HF, but the mechanism for this beneficial cardiovascular effect is not yet clear. [38] It can be a protective compensatory mechanism for reduced body weight in order to enhance appetite and weight gain [39] and/or can be a compensatory response to reduced cardiac output and maladaptive neural and hormonal features of HF. [38] As ghrelin acts as on GHS-R, there is a probability that it is increased in response to GH resistance observed in patients suffering from HF. [14] It has been seen that patients of HF are resistant to the appetite-stimulating effects of ghrelin and that after heart transplantation, there is a decrease in the level of ghrelin, and there is an increase in caloric intake suggesting that ghrelin resistance is resolved after heart transplantation. [14] These states reflects cachexia in HF and weight gain after heart transplantation. [38]

Modulation of sympathetic nervous system

Ghrelin mediates cardioprotective effects by modulating cardiac autonomic nervous activity. [16] However; the precise mechanisms by which ghrelin regulates sympathetic activity are still unclear and needs further investigation. Peripheral ghrelin may act on GHS-R Ia at the cardiac vagal nerve ending, which goes to the nucleus of tractus solitarius and suppresses the renal SNA. [29],[34] Ghrelin can also act directly on the CNS and alter the sensitivity of CNS to other hormones participating in the regulation of sympathetic activity. [40] Endogenously secreted ghrelin reduces mortality, improves heart function and protects the heart from arrhythmias partly by inhibiting sympathetic nervous activity [28] Administration of ghrelin has been shown to lower plasma levels of epinephrine and dopamine and shifts the balance of autonomic nervous activity toward parasympathetic nervous activity. [29]

Insignificant bradycardia after chronic administration of ghrelin [9],[26],[27] suggests that ghrelin offers a beneficial role in the long-term regulation of HR. The role of ghrelin in pathophysiology of hypertension has been recognized widely. Researches show that there is an inverse relation between the amount of circulating ghrelin and arterial stiffness and that circulating level of ghrelin increase after antihypertensive treatment. [23] These evidences suggest that ghrelin is actively involved in the pathophysiology of hypertension and is an independent determinant of stiffness of the arterial wall. [24] However; it has been observed that long-term hypotensive action of ghrelin mediated by the CNS is not as powerful as ghrelin's acute effect on binding protein (BP) regulation as it causes only a modest reduction of BP. This suggests that the effect of ghrelin in the long-term regulation of BP may be reduced by its orexigenic effect. [40]

Inhibits autophagy

Amongst many causes of HF, a gradual decrease in the number of cardiomyocytes is one of the important contributing factors. [41],[42] Studies have suggested that during CHF; cardiomyocyte apoptosis is one of the significant forms of cardiomyocyte loss and can be dangerous at low levels. [43],[44] Therefore, limiting the loss of cardiomyocytes by preventing apoptosis may have implications for the treatment of HF. Ghrelin plays a cardioprotective role against cardiomyopathy through mechanisms that are independent of GHS-R. Ghrelin increases the size of cardiomyocytes, prevents the activation of cardiac fibrosis, reduces autophagy and prolongs their life. [8],[33] It does this by inhibiting the reactive oxygen species and inducing mammalian or mechanistic target of rapamycin that functions in coordinating cell growth and proliferation and also regulates survival of cells. [45] Ghrelin protects the cardiomyocytes against apoptosis, and myocardial injury induced by endoplasmic reticulum stress (ERS) through a GHS-R 1a, Calmodulin-dependent protein kinase kinase (CaMKK) and AMP-activated protein kinase (AMPK) pathway. [18],[40] Subcutaneous administration or preincubation of cardiomyocytes with ghrelin has been shown to stimulate the AMPK. [18],[40],[46] The AMPK on activation is known to inhibit cell proliferation and is involved in autophagy. [47] Preincubation of ghrelin also upregulates ERS markers and cytosine-cytosine-adenosine-adenosine-thymidine/enhancer-binding protein (C/EBP) homologous protein (CHOP). AMPK inhibitors blocks the antiapoptotic actions and ERS inhibitory effects of ghrelin on heart. [40] Ghrelin protects cardiomyocytes against hypoxic injury (induced by COCl 2 ) by inducing autophagy, reducing the expression of NADPH oxidase 1, and increasing the expression and function of antioxidants. [22]

Antiinflammatory effects

Administration of ghrelin reduces nuclear factor-κB (NF-κB) activation and stimulates the macrophages to release antiinflammatory cytokine Interleukin-10 (IL-10) is independent of NF-κB pathway; administration of ghrelin also stimulates the p38 mitogen-activated protein kinases (p38 MAPK), involved in the regulation of release of IL-10 from macrophages. [17],[48]

Protection against ischemia/reperfusion injury

Ghrelin is well-known as a potent activator of release of growth hormone and as growth hormone is involved in tissue regeneration and maintenance of integrity; ghrelin also contributes to the processes of healing and regeneration.

Ghrelin and hexarelin

Hexarelin; a synthetic analogue of ghrelin is superior to ghrelin in improving heart function which can be because of activation of GHS-R Ia and CD36. [28],[29]

Conclusion

Ghrelin is endogenous peptide acting against cardiomyocyte damage. Ghrelin improves cardiac function in rat models of HF though various mechanisms such as modulation of sympathetic nervous system, inhibiting autophagy, antiinflammatory effects and protection against ischemia/reperfusion injury. Ghrelin protects cardiomyocytes against apoptosis induced by ERS through GHS-R1a/CaMKK/AMPK pathways. Ghrelin seems to offer an effective therapeutic target for improving cardiovascular outcomes in experimental models of HF and thus warrants further research.

Meta-analysis of the results was not done as the characteristics of animal models and adopted methodologies of the included studies were quite disparate to coalesce into a single statistical result. In addition, some trials were not randomized and in some trials Ghrelin was administered as a single dose or was administered for a short duration. Also, adverse events and long-term efficacy and safety of ghrelin administration were not available. However; we have explicitly described all the trials in the characteristics of studies table. Though we acknowledge these limitations, the overall data suggests a supporting view that experimental models of rat failure benefit from the effects of ghrelin and that ghrelin can be a tolerable and short-term therapeutic tool in the treatment of HF.

Implications for practice

Exogenous administration with ghrelin may serve as a novel therapeutic drug for various cardiovascular diseases.

Implications for research

Underlying mechanisms governing effect of ghrelin are unexplored. Effects of different analogs of ghrelin in different doses and different rotes warrant further research. Substantiation of hypothesis for the increase in SNA after ghrelin requires further research to study the cellular and molecular pathways involved. More high quality, large, multicentric, human trials are needed to assess the beneficial effects of ghrelin in patients suffering from HF.